Objetivo: O objetivo deste estudo foi analisar a relação e a predominância dos padrões de resposta imune Th1 e Th2 em pacientes com endometriose. Pacientes e Métodos: Entre Fevereiro de 2004 e Abril de 2005 foram avaliadas 98 pacientes divididas em dois grupos de acordo com a presença (Grupo A) ou ausência de endometriose (Grupo B), confirmada histologicamente. Foram coletados sangue periférico e fluido peritoneal de todas as pacientes para a dosagem de interleucinas (IL) 2, 4 e 10, fator de necrose tumoral-alfa (TNF-alfa) e interferon-gama (IFN-gama) por citometria de fluxo. Além da presença da endometriose, foram analisadas a fase do ciclo menstrual, o quadro clinico, o estadiamento, o local de acometimento e a classificação histológica da moléstia. Resultados: Observou-se elevação estatisticamente significante nas concentrações de IFN-gama (mediana de 1,5pg/ml no Grupo A e de 0,4pg/ml no Grupo B, p=0,03) e de IL-10 (mediana de 38,6pg/ml no Grupo A e de 15,7pg/ml no Grupo B, p=0,03) do fluido peritoneal das pacientes com endometriose em relação àquelas sem a doença. As pacientes com endometriose apresentaram alteração estatisticamente significativa na relação das concentrações de IL-4/IFN-gama (p<0,001), IL-4/IL-2 (p=0,006), IL-10/IFN-gama (p < 0,001) e IL-10/IL-2 (p<0,001) do fluido peritoneal, com concentrações mais elevadas da IL-4 e da IL-10, o que reflete o predomínio da resposta Th2 sobre a Th1. Conclusão: Os resultados obtidos permitem concluir que, neste estudo, observou-se elevação de citocinas relativas à resposta imune Th2, denotando haver um predomínio deste padrão de resposta em pacientes com endometriose. / Objective: The objective of this study was to analyze the relation and the predominance of the immune response patterns Th1 and Th2 in patients with endometriosis. Patients and Methods: Between February 2004 and April 2005, 98 patients were evaluated and divided into two groups, according to the presence (Group A) or absence of endometriosis (Group B), confirmed by histology. Peripheral blood and peritoneal fluid were collected from all patients to obtain the concentrations of interleukines (IL) 2, 4 and 10, tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) using flow cytometry. Besides the presence of endometriosis, we analyzed phase of menstrual cycle, clinical complaints, classification, site and histological differentiation of the disease. Results: We observed higher concentrations of IFN-gamma (median of 1.5pg/ml in Group A and 0.4pg/ml in Group B, p = 0.03) and IL-10 (median of 38.6pg/ml in Group A and 15.7pg/ml in Group B, p = 0.03) in peritoneal fluid of patients with endometriosis in relation to those without the disease. Patients with endometriosis presented a significant alteration in IL-4/IFN-gamma (p < 0.001), IL-4/IL-2 (p = 0.006), IL-10/IFN-gamma (p < 0.001) and IL-10/IL-2 (p<0.001) ratio concentrations of peritoneal fluid, with IL-4 and IL-10 predominance, reflecting a Th2 response predominance over the Th1. Conclusion: The results allow concluding that, in this study, it was observed a cytokine elevation related to Th2 immune response, indicating a predominance of this pattern of response in patients with endometriosis.

Asma alérgica é uma inflamação pulmonar crônica mediada por células Th2. A Hipótese da Higiene é a teoria aceita para explicar o aumento das alergias nas últimas décadas e preconiza que a menor exposição dos indivíduos a componentes microbianos prejudica a geração mecanismos imunorregulatórios. Nosso estudo abordou a modulação da resposta alérgica pulmonar induzida por ovalbumina, por cepas de bacilo Calmette-Guérin recombinantes (rBCG) que expressam fragmentos de toxinas bacterianas. Observamos que dependendo do antígeno heterólogo expresso, a imunização intranasal com rBCG pode levar tanto a supressão como a exacerbação da resposta alérgica pulmonar. Demonstramos que tanto em um contexto profilático quanto terapêutico, rBCG é capaz de suprimir os parâmetros alérgicos, e a supressão não envolve o recrutamento de células T regulatórias, é um fenômeno local, está associada a maior produção de IFN-g do que IL-4 e é dependente de IL-12. Estes dados sugerem que a infecção pulmonar por rBCG gera um milieu capaz de bloquear a migração de células Th2 inflamatórias. / Allergic asthma is an atopic disorder mediated by Th2 cells. The Hygiene Hypothesis is the accepted theory to explain the increasing in allergy in recent deacades. It states that modern health care and hygiene practices have led to a reduced exposure to microorganisms components which impairs the generation of immunoregulatory mechanisms. The present study analysed how the intranasal infection with recombinant bacillus Calmette-Guérin (rBCG) strains expressing fragments of bacterial toxins could modulate an allergic pulmonary inflammation induced by ovalbumin. We demonstrated that the rBCG strains could supress or exacerbate the allergic inflammation depending on the expressed heterologous antigen. We analysed the effect of the mycobacterial infection in a prophylactic and in a therapeutical contexts, and we have identified that for both situations the of supression allergic features does not involve the recruitment of regulatory T cells to the lungs, is a local phenomena, is associated with an increased production of IFN-g, and is an IL-12 dependent mechanism. Taken togheter, this data suggest that the rBCG pulmonary infection generates a milieu capable to supress the chemotaxis for Th2 cells, which suppress the establishment of the allergic inflammation in the lungs.

Introdução: O Papilomavírus Humano está muito bem associado com diversos tipos de cânceres humanos, como câncer anogenital e oral. Alguns estudos demonstram que o aparecimento de lesões e a progressão para o câncer estão relacionados ao tipo de resposta imune do hospedeiro. Deste modo, evidências indicam que a resposta imune do hospedeiro tem um papel muito importante para o curso da infecção pelo HPV. Objetivo: Avaliar a resposta imune específica in vitro ao Papilomavírus Humano (HPV) em homens com lesões causadas por HPV e sem lesão por HPV. Material e Métodos: Foram recrutados 31 pacientes e 11 voluntários, que formaram 4 grupos de estudo; sendo 12 pacientes no Grupo A (HIV +/ HPV +); 09 pacientes no Grupo B (HIV-/HPV+); 10 pacientes no Grupo C (HIV+/ HPV-); e 11 indivíduos saudáveis no Grupo D (HIV-/HPV-). Foram realizados ensaios de cultura celular para mensurar a resposta celular específica \"in vitro\" do tipo Th1/Th2/Th17 (INF-y, IL-2, TNFalfa, IL-4, IL-10 e IL-17) sob o estímulo da vacina quadrivalente do HPV (HPV 6, 11, 16 e 18) e à proteína E7 de HPV-16. Resultados: O grupo coinfectado (HIV +/ HPV+) apresentou níveis mais elevados de citocinas, principalmente do perfil Th2, comparando-se com os dados dos demais grupos de estudo. O grupo coinfectado apresentou níveis elevados de IL-6 e IL-10 (Perfil Th2) em relação ao grupo controle (HIV-/HPV-), com significância estatística (p < 0.0001 e p < 0.0001, respectivamente). Conclusão: Foi demonstrada uma elevada produção de citocinas no grupo HPV+/HIV+, sugerindo uma forte imunomodulação pela coinfecção HIV/HPV. Entretanto, novos estudos devem ser realizados para comprovar estes dados. Além de apresentar um perfil essencialmente Th2 do grupo coinfectado, principalmente pelos níveis elevados de IL-6 e IL-10 apresentados, sugerindo que estas duas citocinas possam servir como biomarcadores para persistência viral, uma vez que, os pacientes soropositivos para HIV apresentam maior persistência de HPV, e monitorar a progressão para lesões mais graves / Introduction: Human Papillomavirus is associated with different types of human cancers, such as anogenital and oral cancer. Some studies show that the appearance of lesions and progression to cancer are related to the type of host immune response. Thus, evidence indicates that the host immune response has a role key in the course of HPV infection. Objective: To evaluate the specific immune response in vitro to HPV in men with lesions caused by HPV and without injury caused by HPV. Methods: We recruited 31 patients and 11 volunteers, who formed four groups, with 12 patients in Group A (HIV+/HPV+); 09 patients in Group B (HIV-/HPV+); 10 patients in Group C (HIV+/HPV-) and 11 healthy subjects in Group D (HIV-/HPV-). Cells culture assay was performed to measure the specific immune response \"in vitro\" Th1/Th2/Th17 (IFN-y, IL-2, TNF-alfa, IL-4, IL-10 and IL-17) under the stimulation of quadrivalent HPV vaccine (HPV 6, 11, 16 and 18) and the E7 protein of HPV-16. Results: The coinfected group (HIV+/HPV+) had higher levels of cytokines, especially Th2 profile, compared with data from the other study groups. The coinfected group showed high levels of IL-6 and IL-10 (Th2 profile) compared to the control Group (HIV- /HPV-), with statistical significance (p < 0.0001 and p < 0.0001, respectively). Conclusion: This study demonstrated a high production of cytokines in the coinfected group, suggesting a strong immunomodulation by coinfection HIV/HPV. However, further studies should be conducted to confirm these data. In addition to presenting essentially a Th2 profile, especially by high levels of IL-6 and IL-10 presented, suggesting that these two cytokines may serve as biomarkers for viral persistence, since HIV seropositive patients have a higher persistence of HPV, and monitor the progression to more serious injuries

Objetivo: O objetivo deste estudo foi analisar a relação e a predominância dos padrões de resposta imune Th1 e Th2 em pacientes com endometriose. Pacientes e Métodos: Entre Fevereiro de 2004 e Abril de 2005 foram avaliadas 98 pacientes divididas em dois grupos de acordo com a presença (Grupo A) ou ausência de endometriose (Grupo B), confirmada histologicamente. Foram coletados sangue periférico e fluido peritoneal de todas as pacientes para a dosagem de interleucinas (IL) 2, 4 e 10, fator de necrose tumoral-alfa (TNF-alfa) e interferon-gama (IFN-gama) por citometria de fluxo. Além da presença da endometriose, foram analisadas a fase do ciclo menstrual, o quadro clinico, o estadiamento, o local de acometimento e a classificação histológica da moléstia. Resultados: Observou-se elevação estatisticamente significante nas concentrações de IFN-gama (mediana de 1,5pg/ml no Grupo A e de 0,4pg/ml no Grupo B, p=0,03) e de IL-10 (mediana de 38,6pg/ml no Grupo A e de 15,7pg/ml no Grupo B, p=0,03) do fluido peritoneal das pacientes com endometriose em relação àquelas sem a doença. As pacientes com endometriose apresentaram alteração estatisticamente significativa na relação das concentrações de IL-4/IFN-gama (p<0,001), IL-4/IL-2 (p=0,006), IL-10/IFN-gama (p < 0,001) e IL-10/IL-2 (p<0,001) do fluido peritoneal, com concentrações mais elevadas da IL-4 e da IL-10, o que reflete o predomínio da resposta Th2 sobre a Th1. Conclusão: Os resultados obtidos permitem concluir que, neste estudo, observou-se elevação de citocinas relativas à resposta imune Th2, denotando haver um predomínio deste padrão de resposta em pacientes com endometriose. / Objective: The objective of this study was to analyze the relation and the predominance of the immune response patterns Th1 and Th2 in patients with endometriosis. Patients and Methods: Between February 2004 and April 2005, 98 patients were evaluated and divided into two groups, according to the presence (Group A) or absence of endometriosis (Group B), confirmed by histology. Peripheral blood and peritoneal fluid were collected from all patients to obtain the concentrations of interleukines (IL) 2, 4 and 10, tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) using flow cytometry. Besides the presence of endometriosis, we analyzed phase of menstrual cycle, clinical complaints, classification, site and histological differentiation of the disease. Results: We observed higher concentrations of IFN-gamma (median of 1.5pg/ml in Group A and 0.4pg/ml in Group B, p = 0.03) and IL-10 (median of 38.6pg/ml in Group A and 15.7pg/ml in Group B, p = 0.03) in peritoneal fluid of patients with endometriosis in relation to those without the disease. Patients with endometriosis presented a significant alteration in IL-4/IFN-gamma (p < 0.001), IL-4/IL-2 (p = 0.006), IL-10/IFN-gamma (p < 0.001) and IL-10/IL-2 (p<0.001) ratio concentrations of peritoneal fluid, with IL-4 and IL-10 predominance, reflecting a Th2 response predominance over the Th1. Conclusion: The results allow concluding that, in this study, it was observed a cytokine elevation related to Th2 immune response, indicating a predominance of this pattern of response in patients with endometriosis.

A idade entre o terceiro e o sexto mês de vida é um período peculiar para o estabelecimento da imunidade própria dos bezerros. Nesse intervalo, há susceptibilidade e índice de letalidade à broncopneumonias maiores, provavelmente devido à imaturidade do sistema imunológico pulmonar, quer seja por uma insuficiente resposta, nos primeiros momentos, quer seja por uma resposta citotóxica exagerada, no momento subsequente. A par disso, este trabalho teve o intuito de verificar o momento em que ocorre a maturidade do sistema imunológico, como se comportam os perfis Th1 e Th2 e a existência de uma resposta citotóxica exagerada, durante esta fase de estabelecimento da imunidade ativa dos bezerros. Para tal, estudaram-se as funções de fagocitose e metabolismo oxidativo de leucócitos sanguíneos e broncoalveolares, as classes de imunoglobulinas e citocinas incriminadas nos padrões de resposta linfocitária Th1 e Th2, em 10 bezerros da raça holandesa, sadios, avaliados em sete momentos experimentais, com intervalo quinzenal, entre o terceiro e o sexto mês de vida. Os dados foram submetidos à análise estatística, pela comparação entre as médias ou medianas, confrontadas pelo teste de Anova e Tukey, quando paramétricas, e pelo teste de Kruskal Wallis e Dunn, quando não paramétricas, considerando nível de significância P&le; 0,05 e tendência P&le; 0,10. Os dados que apresentaram dinâmicas semelhantes entre si foram submetidos ao teste de correlação de Pearson. Na região broncoalveolar, observaram-se um aumento progressivo das funções dos macrófagos alveolares, equilíbrio de secreção dos isotipos IgG1 e IgG2 e predominância de citocinas compatíveis com padrão de resposta Th1, até os 150 dias de vida dos bezerros. Aos 165 dias de vida, ocorreram diminuição da função celular, aumento dos títulos de IgG2 e aumento da citocina regulatória IL-10. Aos 180 dias, retornou-se o equilíbrio entre secreção de IgG1 e IgG2, diminuiram os teores de IL-10 e ocorreu tendência a aumento de IL-12, TNF-&#945; e metabolismo oxidativo de macrófagos alveolares, o que permitiu concluir que a resposta imune tem característica própria, nesta faixa etária, e não se torna matura até os seis meses de vida. Apesar de os fagócitos pulmonares já estarem eficientes, a partir dos 135 dias de vida, tornam-se hiperresponsivos aos 150 dias de vida, momento em que gera consequentemente uma resposta regulatória e/ou humoral aos 165 dias de vida, para que aos 180 dias de vida, o equilíbrio entre os perfis Th1 e Th2 seja atingido / The age between the third and sixth month of life is a peculiar period for the establishment of active immunity of calves. There is a greater susceptibility and lethality by bronchopneumonia, probably due to the immaturity of the pulmonary immune system, whether by an insufficient response , in the first moments , whether by an exaggerated cytotoxic response in the subsequent time. So, this work aimed to verify when the maturity of the immune system occurs, how the Th1 and Th2 profiles behave and if there is an exaggerated cytotoxic response during this active phase for immunity of the calves without maternal interference. For this purpose we studied the functions of phagocytosis and oxidative metabolism of blood and bronchoalveolar leukocytes, classes of immunoglobulins and cytokines incriminated in lymphocyte response patterns Th1 and Th2, in 10 holstein healthy calves. They were sampled every fifteen days, during the third until sixth month of life. Data were statistically analyzed by comparing the means or medians, confronted by ANOVA test and Tukey, when the data were parametric, and by Kruskal Wallis and Dunn&#39;s test, when the data were nonparametric, level of significance p &le; 0.05 and trend p &le; 0.10. The data that showed similar dynamics between them were subjected to Pearson correlation test. In bronchoalveolar region, until 150 days of age, the alveolar macrophages functions increased progressively, the IgG1 and IgG2 isotypes secretion showed a balance, and the cytokines profile were compatible with Th1 response. At 165 days of age, there was a decrease of cellular function, an increased of IgG2 titers and the IL-10 secretion, a regulatory cytokine, increased. At 180 days of life, we observed a balance of IgG1 and IgG2 secretion, a decreased of IL-10 levels and a tendency to increase IL-12, TNF-&#945; and alveolar macrophage oxidative metabolism. These results indicated that the calves have an active immune response with particularities for this age group and it does not become mature until six months of life. Despite of the macrophages alveolar are already efficiency from the 135 days of age, they become more reactive at 150 days. After this moment, a regulatory and/or humoral response begins at 165 days of life, as the balance of Th1 and Th2 profiles are reached at 180 days of calves life

Introdução: O Papilomavírus Humano está muito bem associado com diversos tipos de cânceres humanos, como câncer anogenital e oral. Alguns estudos demonstram que o aparecimento de lesões e a progressão para o câncer estão relacionados ao tipo de resposta imune do hospedeiro. Deste modo, evidências indicam que a resposta imune do hospedeiro tem um papel muito importante para o curso da infecção pelo HPV. Objetivo: Avaliar a resposta imune específica in vitro ao Papilomavírus Humano (HPV) em homens com lesões causadas por HPV e sem lesão por HPV. Material e Métodos: Foram recrutados 31 pacientes e 11 voluntários, que formaram 4 grupos de estudo; sendo 12 pacientes no Grupo A (HIV +/ HPV +); 09 pacientes no Grupo B (HIV-/HPV+); 10 pacientes no Grupo C (HIV+/ HPV-); e 11 indivíduos saudáveis no Grupo D (HIV-/HPV-). Foram realizados ensaios de cultura celular para mensurar a resposta celular específica \"in vitro\" do tipo Th1/Th2/Th17 (INF-y, IL-2, TNFalfa, IL-4, IL-10 e IL-17) sob o estímulo da vacina quadrivalente do HPV (HPV 6, 11, 16 e 18) e à proteína E7 de HPV-16. Resultados: O grupo coinfectado (HIV +/ HPV+) apresentou níveis mais elevados de citocinas, principalmente do perfil Th2, comparando-se com os dados dos demais grupos de estudo. O grupo coinfectado apresentou níveis elevados de IL-6 e IL-10 (Perfil Th2) em relação ao grupo controle (HIV-/HPV-), com significância estatística (p < 0.0001 e p < 0.0001, respectivamente). Conclusão: Foi demonstrada uma elevada produção de citocinas no grupo HPV+/HIV+, sugerindo uma forte imunomodulação pela coinfecção HIV/HPV. Entretanto, novos estudos devem ser realizados para comprovar estes dados. Além de apresentar um perfil essencialmente Th2 do grupo coinfectado, principalmente pelos níveis elevados de IL-6 e IL-10 apresentados, sugerindo que estas duas citocinas possam servir como biomarcadores para persistência viral, uma vez que, os pacientes soropositivos para HIV apresentam maior persistência de HPV, e monitorar a progressão para lesões mais graves / Introduction: Human Papillomavirus is associated with different types of human cancers, such as anogenital and oral cancer. Some studies show that the appearance of lesions and progression to cancer are related to the type of host immune response. Thus, evidence indicates that the host immune response has a role key in the course of HPV infection. Objective: To evaluate the specific immune response in vitro to HPV in men with lesions caused by HPV and without injury caused by HPV. Methods: We recruited 31 patients and 11 volunteers, who formed four groups, with 12 patients in Group A (HIV+/HPV+); 09 patients in Group B (HIV-/HPV+); 10 patients in Group C (HIV+/HPV-) and 11 healthy subjects in Group D (HIV-/HPV-). Cells culture assay was performed to measure the specific immune response \"in vitro\" Th1/Th2/Th17 (IFN-y, IL-2, TNF-alfa, IL-4, IL-10 and IL-17) under the stimulation of quadrivalent HPV vaccine (HPV 6, 11, 16 and 18) and the E7 protein of HPV-16. Results: The coinfected group (HIV+/HPV+) had higher levels of cytokines, especially Th2 profile, compared with data from the other study groups. The coinfected group showed high levels of IL-6 and IL-10 (Th2 profile) compared to the control Group (HIV- /HPV-), with statistical significance (p < 0.0001 and p < 0.0001, respectively). Conclusion: This study demonstrated a high production of cytokines in the coinfected group, suggesting a strong immunomodulation by coinfection HIV/HPV. However, further studies should be conducted to confirm these data. In addition to presenting essentially a Th2 profile, especially by high levels of IL-6 and IL-10 presented, suggesting that these two cytokines may serve as biomarkers for viral persistence, since HIV seropositive patients have a higher persistence of HPV, and monitor the progression to more serious injuries

Asma alérgica é uma inflamação pulmonar crônica mediada por células Th2. A Hipótese da Higiene é a teoria aceita para explicar o aumento das alergias nas últimas décadas e preconiza que a menor exposição dos indivíduos a componentes microbianos prejudica a geração mecanismos imunorregulatórios. Nosso estudo abordou a modulação da resposta alérgica pulmonar induzida por ovalbumina, por cepas de bacilo Calmette-Guérin recombinantes (rBCG) que expressam fragmentos de toxinas bacterianas. Observamos que dependendo do antígeno heterólogo expresso, a imunização intranasal com rBCG pode levar tanto a supressão como a exacerbação da resposta alérgica pulmonar. Demonstramos que tanto em um contexto profilático quanto terapêutico, rBCG é capaz de suprimir os parâmetros alérgicos, e a supressão não envolve o recrutamento de células T regulatórias, é um fenômeno local, está associada a maior produção de IFN-g do que IL-4 e é dependente de IL-12. Estes dados sugerem que a infecção pulmonar por rBCG gera um milieu capaz de bloquear a migração de células Th2 inflamatórias. / Allergic asthma is an atopic disorder mediated by Th2 cells. The Hygiene Hypothesis is the accepted theory to explain the increasing in allergy in recent deacades. It states that modern health care and hygiene practices have led to a reduced exposure to microorganisms components which impairs the generation of immunoregulatory mechanisms. The present study analysed how the intranasal infection with recombinant bacillus Calmette-Guérin (rBCG) strains expressing fragments of bacterial toxins could modulate an allergic pulmonary inflammation induced by ovalbumin. We demonstrated that the rBCG strains could supress or exacerbate the allergic inflammation depending on the expressed heterologous antigen. We analysed the effect of the mycobacterial infection in a prophylactic and in a therapeutical contexts, and we have identified that for both situations the of supression allergic features does not involve the recruitment of regulatory T cells to the lungs, is a local phenomena, is associated with an increased production of IFN-g, and is an IL-12 dependent mechanism. Taken togheter, this data suggest that the rBCG pulmonary infection generates a milieu capable to supress the chemotaxis for Th2 cells, which suppress the establishment of the allergic inflammation in the lungs.

En 2014 la Tuberculose (TB) à dépassé le VIH comme la principale cause de décès par maladie infectieuse dans le monde soulignant le besoin urgent de développer un vaccin plus efficace contre cette maladie. Le candidat vaccin contre la TB, ID93/GLA-SE, dévéloppé à l’Infectious Disease Research Institute (IDRI), est aujourd’hui en essai clinique de phase IIa et a montré des résultats pré-cliniques et cliniques promettants. Dans de modèle murin de TB, ce vaccin induit une forte réponse TH1, considérée comme centrale dans la protection contre la TB, et la production d’IgG2 par les lymphocytes B. Néanmoins, les mécanismes d’action de GLA-SE sont encore peu connus.L’objectif principal de cette thèse est donc d’élucider les méchanismes clés qui relient les réponses innées et adaptatives induites par cet adjuvant dans le modèle murin. Un objectif secondaire est d’établir un modèle murin de rechute de TB après traitement et d’évaluer l’utilisation d’ID93/GLA-SE en tant que vaccin immuno-thérapeutique et sa capacité à réduire les taux de rechute dans ce modèle. L‘ensemble de ce travail nous a permis de mieux comprendre les mécanismes impliqués dans la réponse immunitaire adaptative induite par GLA- SE et de montrer la capacité de ID93/GLA- SE a être utilisé comme un vaccin thérapeutique contre la tuberculose dans le but de réduire les taux de rechute post-thérapeutiques. / In 2014 tuberculosis (TB) surpassed HIV as the leading cause of death by an infectious disease worldwide emphasizing the urgent need to develop a more effective vaccine against this airborne disease. The Infectious Disease Research Institute (IDRI) TB candidate vaccine ID93/GLA-SE is currently undergoing a Phase IIa clinical trial and has shown promising preclinical and clinical results. In murine models of TB this vaccine drives a strong CD4 TH1 response, which is thought to be important for protection against TB, and an IgG2c skewed B cell response. However, little is known about the cellular and molecular events that drive GLA-SE adjuvanticity.To that end, the main objective of my thesis was to elucidate the key mechanisms that connect innate and adaptive immune responses elicited by this adjuvant in the murine model. A secondary objective was to evaluate the possibility of using ID93/GLA-SE as adjunct therapy to existing antibiotic treatments to reduce relapse rates after TB treatment.Collectively the results obtained during this research project and thesis broaden our knowledge and our current understanding of the mechanisms involved in the adaptive immune response induced by GLA-SE and show the capacity of ID93/GLA-SE to be used as a therapeutic vaccine against TB to reduce post-therapeutic relapse rates.

Das Bestreben, den Aufbau, die Funktion sowie die Entwicklung des Immunsystems zu verstehen, steht schon lange Zeit im Zentrum des Interesses vieler Forschungsarbeiten, insbesondere um auf Grundlage der gewonnenen Erkenntnisse neue Behandlungsansätze für immunologisch relevante Krankheitsbilder zu entwickeln. Stillen könnte ein wichtiger Faktor sein, der bei der Entwicklung und Differenzierung von T-Zell-Subpopulationen und Zytokinmustern im Säuglings- und Kindesalter eine bedeutende Rolle spielt. Die Zielsetzung der hier vorgelegten Promotionsarbeit war es, den potentiellen Effekt des Faktors Stillen auf die Entwicklung, die Verteilung und die Differenzierung von Zell-populationen sowie die Expression von Zytokinen bei gesunden Kindern zu untersuchen. Dies geschah insbesondere im Hinblick auf einen möglicherweise vorhandenen Shift der relativen Verteilung der TH1- und TH2-Zytokinen, da in retrospektiven Kohortenstudien bereits gezeigt werden konnte, dass gestillte Kinder eine geringere Anfälligkeit gegenüber schwerwiegenden bakteriellen Infektionen (BACHRACH ET AL., 2003) sowie einer verminderten Inzidenz von Autoimmunerkrankungen (KOLETZKO ET AL., 1989; PISACANE ET AL., 1994) aufweisen. Die Studienkohorte bestand aus 196 gesunden Kindern im Alter zwischen 26 Tagen und 12 Jahren und 352 Tagen. Diese wurde in vier Altersgruppen unterteilt (<1, 1- <3, 3- <6 und 6-<13 Jahre) und mittels eines Fragebogens im Hinblick auf ein möglicherweise vorhandenes Bias bezüglich exogener Einflussfaktoren wie Impfungen, Nikotinexposition (FELESZKO ET AL., 2006) und allergische Erkrankungen in der Familie (HRDÝ ET AL., 2010), die in diesem Zusammenhang diskutiert werden, überprüft. Dabei zeigten sich keine signifikanten Unter-schiede zwischen den Gruppen. Alle immunologischen Parameter wurden in peripherem, heparinisiertem Blut ermittelt. Zunächst wurde mittels Durchflusszytometrie (FACS) eine Phänotypisierung, anhand von antikörpermarkierten Oberflächenantigenen der T-, B- und NK-Zellpopulationen („Immun-status“), durchgeführt. Des Weiteren wurden die mononuklearen Zellen des peripheren Blutes (PBMC) mittels PMA und Ionomycin stimuliert und die Zytokinsekretion durch Brefeldin blockiert. Durch FACS-Analyse wurde die nach 20-24 stündiger Anregung in der Kultur vorhandene intrazellulärer Zytokinexpression von IL2, IFNγ, TNFα, IL4, IL10, TGFβ und IL17 in den T-Zellpopulationen ermittelt. In einem zweiten Schritt wurde der Quotient aus IFNγ und IL4 berechnet, um das Verhältnis zwischen TH1 und TH2 zu analysieren. Das Datenmaterial zeigt die Entwicklung der T-Zell-Subpopulationen mit dem Alter. Junge Kinder zeigen eine durch regulatorische T-Zellen (Treg) bzw. TH0-Zellen vorherrschende TGFβ und IL2-Expression, während ältere Kinder das gesamte Repertoire an TH1 (IFNγ, TNFα) und TH2-Zytokinen (IL4), insbesondere durch T-Gedächtniszellen, exprimieren. Diese Ergebnisse bestätigten bereits zuvor beschriebene – vom Faktor Stillen unabhängige – altersabhängige Veränderungen der Zellpopulationen und der Zytokinexpression. Aus diesem Grund konnte von einer „normalen Verteilung“ der Studienkohorte ausgegangen werden. Zwischen gestillten und nicht-gestillten Kindern hat sich gezeigt, dass sich die Größe und das Verhältnis der übergeordneten Zellpopulationen (T-Helferzellen, zytotoxische T-Zellen) sowie die Reifung der T-Zellen (naive T-Zellen, T-Gedächtniszellen) bezogen auf das Alter nicht unterscheiden. Hingegen zeigt der Faktor Stillen in der Tat einen Einfluss auf die TH1/TH2-Balance. Bei gestillten Kindern lässt sich eine stärkere Gewichtung in Richtung der TH2-Zytokine feststellen (niedrigere IFNγ/IL4-Ratio), so dass davon auszugehen ist, dass Stillen einen so genannten TH2-Shift induziert. Dieses gegenüber nicht-gestillten Kindern „verschobene“ Gleichgewicht bleibt bis zur Altersgruppe der 6-13 Jährigen konstant. Gestillte Kinder haben zudem, im Vergleich zu Formula-ernährten Kindern, ein höheres Vermögen TH1-Zytokine wie TNFα und IFNγ zwischen dem dritten und sechsten Lebensjahr zu bilden. Diese Daten entsprechen nicht einem vorherrschenden TH2-Muster und einer Allergiedisposition, aber sie können die geringere Inzidenz von bakteriellen Infektionen im Vorschulalter (TH1-Antworten benötigt) und von TH1-vermittelten Autoimmunerkrankungen bei gestillten Kindern erklären. Aufgrund der Ergebnisse wird deutlich, dass Muttermilch einen bedeutenden Einfluss auf das Immunsystem hat. Insbesondere konnte gezeigt werden, dass der Einfluss der Muttermilchernährung über den eigentlichen Zeitraum des Stillens hinausreicht. Eine Prägung des Immunsystems durch die Muttermilch, während der frühen Kindheit, erscheint deshalb sehr wahrscheinlich. Diese Ergebnisse sollten selbstverständlich durch weiterreichende Studien, die eventuell vorhandene weitere Störgrößen wie genetische Prädispositionen oder auch Umwelttoxine einschließen, verifiziert werden. Zudem wäre es von Interesse welche in der Muttermilch enthaltene Stoffe zu diesem TH2-Shift beitragen oder ob es sich bei den Einflussfaktoren vielmehr um in der Muttermilch nicht enthaltene Stoffe handelt, die in Formula-Nahrung enthalten sind, wie zum Beispiel Kuhmilchantigene. Schlussendlich bleibt festzuhalten, dass Muttermilch erwiesene positive Vorteile mit sich bringt, wie geringere Infektionsraten (Atemwegsinfektionen, Otitis media) und eine Risiko-verminderung für bestimmte Erkrankungen (Diabetes mellitus Typ1, Morbus Crohn, Multiple Sklerose) im späteren Leben. Die Ergebnisse dieser Arbeit können bestätigen, dass das Stillen mit Muttermilch tatsächlich einen Einfluss auf die Entwicklung des individuellen Immunsystems hat, der auch nach dem Abstillen weiter anhält. Eine wichtige Rolle kann diese Erkenntnis bei der Beratung werdender Mütter spielen, gerade in Hinblick auf ein gegebenenfalls erhöhtes endogenes familiäres Risiko für beispielsweise Autoimmunerkrankungen. Folgearbeiten sind sicher wünschenswert, um den pathophysiologischen Hintergrund dieser beobachteten Daten besser zu verstehen. / The aspiration to understand the construction, the function as well as the development of the immune system is for a long time in the centre of the interest of many research projects, in particular to develop new attempts of treatment for immunological relevant clinical pictures on basis of the won knowledge. Breast-feeding could be an important factor which plays an important role by the development and differentiation of T-cell subsets and cytokine profiles in infancy and childhood. The objective of the doctorate work presented here was to examine the potential effect of the factor breast-feeding for the development, the distribution and the differentiation of cell populations as well as the expression of cytokines with healthy children. This happened in particular in view of a possibly available Shift of the relative distribution of the TH1 and TH2 cytokines, because in retrospective cohort studies could be already demonstrated, that breastfed children show a lower susceptibility for serious bacterial infections (BACHRACH ET AL.,2003) as well as a decreased incidence of autoimmune illnesses (KOLETZKO ET AL.,1989; PISACANE ET AL.,1994). The study cohort existed of 196 healthy children at the age between 26 days and 12 years and 352 days. This was divided into four age groups (<1, 1-<3, 3-<6 and 6-<13 years) and by means of a questionnaire in view of a possibly available Bias with regard to exogenous factors of influence like vaccinations, nicotine exposition (FELESZKO ET AL.,2006) and allergic illnesses in the family (HRDÝ ET AL.,2010) which are discussed in this connection, checks. Besides, no significant differences appeared between the groups. All immunological parametres were determined in peripheral, heparinised blood. First a phenotype determination, on the basis of antibody-marked surface antigens of the T-, B- and NK-cell populations ("immune status"), was carried out by means of flow cytometry (FACS). Besides the mononuclear cells of the peripheral blood (PBMC) were stimulated by means of PMA and Ionomycin and the cytokine secretion was blocked by Brefeldin. By FACS analysis the cytokin expression of IL2, IFN γ, TNF α, IL4, IL10, TGF β and IL17 in the T-cell subpopulations was determined. In the second step the quotient from IFN γ and IL4 was calculated to analyse the relation between TH1 and TH2. The data material shows the development of the T-cell subsets with the age. Young children show one by regulatory T-cells (Treg) or TH0-cells prevailing TGF β and IL2 expression, while older children can produce the whole repertoire of TH1-(IFN γ, TNF α) and TH2-cytokines (IL4), in particular by memory T-cells. These results already confirmed before described – from the factor breast-feeding independent – changes of the cell populations and the cytokine production dependent on age. That's why could be gone out from a „normal distribution“ of the study cohort. Between breastfed and non-breastfed infants has appeared that the size and the relation of the higher cell populations (T-helper cells, zytotoxic T-cells) as well as the maturation of the T- cells (naive T-cells, memory T-cells) did refer to the age make no distinction. Indeed, however, breastfeeding shows an influence on the TH1/TH2-balance. Breastfed children show a stronger weighting in the direction of the TH2-Zytokine (lower IFN γ/IL4-Ratio), so that is to be assumed from the fact that breast-feeding induces a so-called TH2-Shift. These towards non-breastfed children "postponed" balance remains up to the age group of the 6-13 year-old steady. Besides, breastfed children have a higher ability, in comparison to Formula-fed children, to produce TH1-Zytokine like TNF α and IFN γ between the third and sixth year of life. These data do not correspond to a prevailing TH2 pattern and an allergy arrangement, but they can explain the lower incidence of bacterial infections in the pre-school age (TH1-answers needed) and from TH1-provided auto immune illnesses with breastfed children. On account of the results becomes clear that breastmilk has an important influence on the immune system. In particular it could be shown that the influence of breastmilk stretches beyond the real period of the breast-feeding. Therefore, an imprinting of the immune system by breastmilk, during the early childhood, seems very likely. These results should be verified of course by the handing on studies which enclose, perhaps, available other sturgeon dimensions like genetic prearrangements or also environmental toxins. Besides, it would be of interest which ingredients of breastmilk to this TH2-Shift contribute or whether it concerns with the factors of influence rather materials not contained in the mother's milk which are included in Formula food, as for example cow's milk antigens. Finally remains to stick that mother's milk brings proved positive advantages with itself, how lower infection rates (breath way infections, Otitis media) and a risk decrease for certain illnesses (diabetes mellitus Typ1, Morbus Crohn, multiple sclerosis) in the later life. The results of this work can confirm that the breast-feeding with mother's milk really has an influence on the development of the individual immune system. An important role can play this knowledge with the consultation of mothers, just in view of an endogenous informal risk raised if necessary for example auto immune illnesses. Subsequent works are surely desirable to understand the pathophysiological background of these observed data better.

RESUME Depuis leur découverte en 1973 par Steinman et Cohn, le rôle des cellules dendritiques dans l’initiation des réponses immunitaires a largement été documenté. En effet, les cellules dendritiques constituent les cellules présentatrices d’antigènes professionnelles capables de détecter des molécules microbiennes et inflammatoires afin d’activer le système immunitaire. Outre leur implication dans l’induction des réponses immunes, de plus en plus d’études suggèrent que les cellules dendritiques interviennent dans le contrôle des réponses immunitaires via la sécrétion de cytokines anti-inflammatoires et/ou l’activation ou l’induction de lymphocytes T régulateurs. Ceux-ci incluent les cellules T régulatrices issues naturellement du thymus et les cellules T régulatrices induites en périphérie. Des résultats obtenus au sein de notre laboratoire ont mis en évidence l’importance des cellules T régulatrices dans le contrôle des réponses de type Th1 induites à l’aide de cellules dendritiques matures chargées avec des antigènes étrangers. Nous avons, dès lors, étudié le rôle du récepteur CTLA-4 exprimé constitutivement à la surface des cellules T régulatrices dans le contrôle des réponses immunitaires induites à l’aide de cellules dendritiques matures et dans un modèle d’inflammation intestinale. L’injection d’anticorps anti-CTLA-4 induit in vitro et in vivo une inhibition de la production d’IFNγ et protège les souris de la colite pro-Th1 induite par l’instillation de TNBS. Cette protection corrèle étroitement avec l’induction de lymphocytes T régulateurs exprimant fortement la molécule ICOS et sécrétant de l’interleukine 10. De plus, nos résultats suggèrent que l’interleukine 10 et l’indoléamine 2, 3 dioxygénase seraient impliquées dans la fonction régulatrice des lymphocytes T ICOShigh. Nous avons également analysé les mécanismes impliqués dans le contrôle des réponses de type Th1 par les lymphocytes T régulateurs naturels. Nos résultats suggèrent une régulation différente des réponses Th1 en présence et en absence de cette population régulatrice. En effet, les réponses Th1 sont dépendantes de l’interleukine 12 en présence de lymphocytes T régulateurs naturels, alors qu’en leur absence, la molécule CD70 est requise. En conclusion, nos résultats suggèrent que les lymphocytes T régulateurs naturels et induits contrôlent les réponses immunes de type Th1. Au cours de ce travail, nous avons mis en évidence des stratégies distinctes par lesquelles ces deux populations régulatrices contrôlent la réponse immune. Ces résultats complètent la compréhension des mécanismes de régulation du système immunitaire et ouvrent de nouvelles perspectives d’approche immunothérapeutique.